Tumor cell-specific homozygous deletions coinciding at a particular genetic location may indicate the inactivation of a nearby tumor suppressor gene. Forty-six human cancer cell lines of prostate, pancreatic, lung, liver, and colon origin were screened for homozygous deletions of 139 expressed sequence tag (EST) and sequence-tagged site (STS) loci spanning the entire short arm of chromosome 8. Only one Southern blot-verified homozygous deletion was detected in this set of cell lines. The deletion, in pancreatic tumor cell line MIA-PaCa-2, encompassed two screening loci, D8S549 and D8S1992, and overlapped another previously described homozygous deletion of band 8p22 in a metastatic prostate cancer specimen. Both deletions entirely removed the candidate tumor suppressor gene N33. These data define a consensus homozygous deletion region in chromosome band 8p22.